Nonsteroidal anti-inflammatory drugs (NSAIDs1) are widely used for the treatment of pain, inflammation, and fever (Donnelly and Hawkey, 1997; Jouzeau et al., 1997). The mechanism of action of many of these drugs (e.g., meloxicam, lornoxicam, tenoxicam, and indomethacin) is thought to involve the inhibition of COX, a hemeprotein that has been shown to convert arachidonic acid to proinflammatory prostaglandins and their subsequent metabolic products (Wu, 1998). COX behaves as an endoperoxide synthetase, catalyzing the formation of the cyclic endoperoxide prostaglandin G2 from the unesterified precursor fatty acid, and as a peroxidase that converts prostaglandin G2 to prostaglandin H2. Prostaglandin H2 is then converted to other products (e.g., prostaglandin I2 and thromboxane A2) via the action of prostacyclin synthetase and thromboxane synthetase.

COX has been shown to exist in two forms (COX-1 and COX-2). COX-1 is thought to carry out “housekeeping” functions (e.g., cytoprotection of the gastric mucosa and platelet aggregation) and catalyzes the production of prostaglandins under normal physiological conditions. By comparison, COX-2 is normally undetectable in most tissues and is inducible by cytokines, endotoxins, and tumor promotors (Donnelly and Hawkey, 1997; Jouzeau et al., 1997). Since the products of COX-1 are cytoprotective, selective inhibition of COX-2 is anticipated to reduce inflammation without the gastrointestinal side effects characteristic of NSAIDs currently in use (Donnelly and Hawkey, 1997; Jouzeau et al., 1997; Riendeau et al., 1997; Wu, 1998). Therefore, much attention has been focused on the design of potent and selective COX-2 inhibitors (Leblanc et al., 1995, 1999; Penning et al., 1997; Janusz et al., 1998). One such example is etoricoxib (Fig.1), which is under investigation for the treatment of osteoarthritis and rheumatoid arthritis. The drug has been shown to be a potent and selective inhibitor of COX-2 in vitro and exhibits pharmacological activity in vivo (Friesen et al., 1998;Riendeau et al., 2000). In an earlier study, the in vitro metabolites of etoricoxib [5-chloro-3-(4-methanesulfonylphenyl)-6′-methyl-[2,3′]-bipyridinyl] were characterized, and preliminary studies with recombinant P450s suggested that multiple P450s were involved in the oxidative metabolism of the drug (Chauret et al., 2001). Although etoricoxib is a low clearance compound (0.8 ml/min/kg), with high oral bioavailability (>80%), it has been shown to undergo extensive biotransformation (>90% of the dose) to metabolites that are detected in urine and feces (R. Halpin and N. Agrawal, unpublished observations). Because it is likely that etoricoxib will be administered with other drugs in a clinical setting, we sought to study the in vitro NADPH-dependent metabolism of [14C]etoricoxib in native human liver microsomes and to identify the P450 forms involved in its metabolism. We also wanted to determine the relevance of the in vitro data to the situation in vivo by determining the metabolite profile of etoricoxib in human urine.

Incubation of Etoricoxib with Native Human Liver Microsomes.

In vitro incubations were performed at 37°C in a Dubnoff shaking water bath, using 12 × 75 mm borosilicate glass disposable culture tubes (Machinist et al., 1995; Rodrigues et al., 1996). Briefly, the final assay volume was 0.25–1.0 ml and consisted of the following: 0.1 M potassium phosphate buffer (pH 7.4), magnesium chloride (5 mM), NADP+ (1.0 mM),d-glucose 6-phosphate (10 mM), d-glucose 6-phosphate dehydrogenase (Sigma Type VII, from baker's yeast, 2.0 units/ml), microsomal protein (0.5–2.5 mg/ml), and [14C]etoricoxib (1–1300 μM) dissolved in acetonitrile (<1% v/v, final volume in assay). The reactions were initiated by addition of the NADPH generating system after a 3-min preincubation period (37°C, open to air) and then stopped by addition of 1.5% (v/v) glacial acetic acid in acetontrile:methanol (2:1, v/v). In each case, the sample was centrifuged (2000g, 10 min), and the supernatant was decanted into a clean tube. After evaporation to dryness under nitrogen at 37°C, the dried residue was reconstituted in 0.2 ml of acetonitrile (30%, v/v). An aliquot of sample (0.1 ml) was analyzed by HPLC with radiometric detection.

P450 Selective Inhibitors.

Inhibition studies with P450 form selective chemical inhibitors were carried out at a final [14C]etoricoxib concentration of 5 μM (<Km) and 200 μM (∼Km). Where possible, mechanism-based inhibitors (e.g., troleandomycin and furafylline), relatively high affinity reversible inhibitors (Ki ≤ 1.0 μM), or cosubstrates (Km ≤5.0 μM) were used. Where appropriate, the final concentration of the inhibitor (cosubstrate) exceeded (≥ 10-fold) its apparentKi (Newton et al., 1995; Bourrie et al., 1996; Rodrigues et al., 1996). This ensured maximal inhibition (>80%) of each P450 form ([I]/Ki≥10; [S] ≤ Km). In addition, the inhibitors were dissolved in acetonitrile:water (50:50, v/v), in which case the final concentration of acetonitrile in the incubations was kept to a minimum (≤1.0% v/v).

Incubations with [14C]etoricoxib (50 μM) were conducted at 37°C, in 1.5 ml polypropylene centrifuge tubes (final volume of 0.25 ml to 0.6 ml), and were conducted as described for liver microsomes, except that the molarity of phosphate buffer was decreased to 10 mM. Samples were preincubated for 5 min, after which time the reaction was started with the addition of rapidly thawed (37°C) microsomal protein (0.5 mg/ml final concentration). In all cases, the reactions were allowed to proceed for the maximum period of linearity (CYP3A4 + b5, 20 min; CYP1A2, 80 min; CYP2A6, 40 min; CYP2C9*1, 10 min; CYP2C8, 160 min; CYP2C19, 10 min; and CYP2D6, 10 min).

For all P450 proteins tested, the reaction rates (pmol/h/pmol of P450) were normalized (pmol/h/pmol · pmol of P450/mg) with respect to the corresponding nominal (mean) specific content of each P450 in native human liver microsomes (data provided by GENTEST Corp.; CYP3A4, 108 pmol/mg; CYP1A2, 45 pmol/mg; CYP2A6, 68 pmol/mg; CYP2C9, 96 pmol/mg; CYP2C8, 64 pmol/mg; CYP2C19, 19 pmol/mg; and CYP2D6, 10 pmol/mg). The normalized rates (pmol/h/mg) were then added, and the normalized rate for each P450 was expressed as the percentage of the total normalized rate (Rodrigues, 1999).

Immunoinhibition Studies.

[14C]Etoricoxib (50 μM) was incubated with a native human liver microsomes (pool of samples HHM-0288, HHM-0232, and HHM-0253) as described previously (0.24 mg protein/ml; 146 pmol of P450/ml). Incubations (final assay volume of 0.25 ml) were performed in the presence of increasing amounts (0–18 mg IgG/nmol of P450) of preimmune sera or anti-CYP3A4 peptide polyclonal antibodies (Wang and Lu, 1997).

HPLC.

Etoricoxib and its metabolites were separated on a reverse-phase C8 (Zorbax Eclipse XDB-C8, 4.6 × 250 mm, 5 μm) column using a Hewlett-Packard HP1100 liquid chromatography system with the column oven temperature set at 40°C. The mobile phase consisted of (A) 20% acetonitrile:80% ammonium acetate (25 mM, pH 7–7.4), and (B) acetonitrile and was programmed to go from 0 to 30% B over 30 min at a flow rate of 1 ml/min. The effluent was monitored by photodiode array detection at 236 and 280 nm, and by an on-line radiometric detector (β-RAM, INUS Systems, Inc., Tampa, FL) using a 3 ml/min flow rate for the scintillation cocktail. Under these conditions, the retention times (±0.5 min) of etoricoxib, M2 (6′-methyl hydroxy metabolite), M1 (1′-N-oxide metabolite), andM3 (6′carboxy metabolite) were 26, 17, 14, and 9 min, respectively.

LC/MS.

Metabolites of etoricoxib were identified by electrospray LC-MS/MS analysis using the Finnigan LCQ mass spectrometer. The spray voltage was held at 4.1 kV, and the capillary temperature and voltage were set at 200°C and 6.0 V, respectively. Samples (microsomal and urine extracts) were dissolved in 30% aqueous acetonitrile and introduced into the mass spectrometer via a Zorbax eclipse XDB-C8 column. A gradient of 25 mM ammonium acetate and acetonitrile was used as the mobile phase. In positive-ion mode MS, the 6′-hydroxymethyl metabolite (M2), yielded an MH+ ion (35Cl) atm/z 375, which upon MS/MS analysis produced intense fragment ions at m/z 357 (MH+-H2O) and 278. Similarly, the N-oxide metabolite (M1) yielded an MH+ ion at m/z 375 and produced ions at m/z 357, 296, and 278. The MS/MS spectrum (precursor m/z 389, MH+) of the carboxyl metabolite (M3) exhibited intense ions at m/z 343, 328, and 280. Both the product ion spectra and HPLC retention times of the microsomal and urinary metabolites were similar to those obtained from samples of authentic standards and confirmed earlier findings (Chauret et al., 2001).

Analysis of Human Urine.

Six healthy male subjects received an i.v. (25 mg, 100 μCi) dose of [14C]etoricoxib. The dose was administered in citrate-buffered saline (pH 3.6, 0.75 mg/ml) as a 15-min infusion. Urine (0–2, 2–4, 4–6, 6–9, 9–12, 12–18, and 18–24 h postdose) was collected and stored frozen at −20°C. Prior to analysis, the urine was thawed at room temperature, and aliquots from each time interval were combined in proportion to their respective volumes to produce a representative 0–24 h sample for each subject. Aliquots (0.2 ml) were taken for liquid scintillation counting. A second aliquot (5 ml) was transferred to glass centrifuge tubes, and trifluoroacetic acid (15 μl) was added to acidify the samples to approximately pH 3. Acetonitrile (20 ml) was added with mixing, and particulate material was removed by centrifugation (2000 g, 10 min). The supernatants were transferred to 50-ml glass tubes, and solvent was removed in a centrifugal vacuum concentrator (Speed-Vac, Savant, Holbrook, NY). The residues were reconstituted in mobile phase and analyzed by HPLC with radiometric detection as described previously.

Results

P450-Dependent Metabolism of Etoricoxib.

A typical radiochromatograph of the supernatant following incubation of [14C]etoricoxib with NADPH-fortified human liver microsomes is presented in Fig. 2A. After incubation, one major metabolite peak was observed (retention time of 17 min), which coeluted with authentic M2 standard and was identified by LC/MS as the 6′-methyl hydroxy metabolite of etoricoxib. Omission of the NADPH generating system completely abolished the hydroxylation reaction by human liver microsomes, indicating that metabolite formation was enzymatic and NADPH-dependent. By comparison, low levels of the 1′-N-oxide metabolite (M1) were detected in the presence of the NADPH generating system (Fig. 2A). In addition, no secondary metabolism was detected, although the 6′-carboxy metabolite (M3) accounted for the majority of the radioactivity in human urine (Fig. 2B) and feces (R. Halpin unpublished observations).

In a panel of human liver microsomes (n = 15 different organ donors), the interindividual variability in the rate of 6′-methyl hydroxylation ranged from 3.5-fold at a final etoricoxib concentration of 200 μM (range 105–372 pmol/min/mg; mean of 203 pmol/min/mg) to 9.5-fold at a final etoricoxib concentration of 5 μM (range 3.2–18.5 pmol/min/mg; mean of 9.5 pmol/min/mg).

Summary of the apparent kinetic parameters for the hydroxylation of etoricoxib by native human liver microsomes

The in vitro intrinsic clearance (Vmax/Km ratio) was scaled, with respect to yield of microsomal protein and liver weight, and yielded a value of 3.1 to 9.7 ml/min/kg of b.wt. as the formation clearance of M2. Similarly, the in vivo intrinsic clearance was estimated to be 8.3 ml/min/kg, using i.v. data obtained from six subjects.

Correlation Studies.

Etoricoxib (5 and 200 μM) hydroxylation was significantly correlated (r = 0.80–0.89; p < 0.001;n = 15) with CYP3A4/5-selective testosterone 6β-hydroxylase activity in the bank of liver microsomes. By comparison (Table 2), the correlation with 7-ethoxyresorufin O-deethylase (CYP1A2), (S)-(+)-mephenytoin 4′-hydroxylase (CYP2C19), tolbutamide methyl hydroxylase (CYP2C9), chlorzoxazone 6-hydroxylase (CYP2E1), and bufuralol 1′-hydroxylase (CYP2D6) activity was relatively weak (r ≤ 0.47). It is important to note that, while the correlations with CYP2A6-selective coumarin hydroxylase (r = 0.71; p < 0.01) and CYP2C8-selective Taxol 6-hydroxylase (r = 0.72;p < 0.01) activities are significant, both activities correlated with the rates of testosterone 6β–hydroxylation (r ≅ 0.79; p < 0.001) and, because of coregression, correlation with etoricoxib hydroxylation would be expected. Moreover, correlation of etoricoxib hydroxylase activity with these enzymes did not improve in the presence of CYP3A4 monoclonal antibody (data not shown). Overall, the data indicate that CYP3A subfamily member(s) play a major role in the formation of M2over a relatively wide etoricoxib concentration range (5–200 μM). In agreement, the correlation of M2 formation at two different concentrations of etoricoxib (5 μM versus 200 μM) was significant (r = 0.81; p < 0.001), also suggesting that the same P450 form(s) catalyzed the reaction over the etoricoxib concentration range studied.

Correlation of various P450-selective monooxygenase activities with the hydroxylation of etoricoxib (5 and 200 μM) in a panel of human liver microsomes

Chemical Inhibition Studies.

Ketoconazole (2.0 μM) and troleandomycin, both selective inhibitors of CYP3A in native human liver microsomes, were shown to effectively decrease (65–70%) the rate of M2 formation at two concentrations (5 and 200 μM) of etoricoxib (Fig.4). By comparison, chemical inhibitors selective for other P450s were relatively weak, although some inhibition (∼10%) was observed in the presence of furafylline (CYP1A2-selective), sulfaphenazole (CYP2C9-selective), or quinidine (CYP2D6-selective). In agreement with correlation studies, these data suggested that the 6′-methyl hydroxylation of etoricoxib was primarily catalyzed by member(s) of the CYP3A subfamily, although other P450s (∼10% each), such as CYP1A2, CYP2D6, and CYP2C9 also played a role. When sulfaphenazole (5 μM), ketoconazole (2 μM), and quinidine (10 μM) were coincubated, M2 formation was inhibited by approximately 84 ± 5.7% (data not shown), suggesting that CYP3A, CYP2C9, and CYP2D6 accounted for the majority of the oxidative (NADPH-dependent) metabolism of etoricoxib in native human liver microsomes.

In a separate series of experiments, the inhibitory effect of troleandomycin was confirmed with the bank of human liver microsomes (Fig. 5). Inhibition in the presence of troleandomycin (mean ± S.D. = 68 ± 13.4; n= 16 subjects), which varied from 40% (subject 24) to 90% (subject 2), correlated (r = 0.87; p < 0.001) with the levels of CYP3A (testosterone 6β-hydroxylase) activity (data not shown). Despite a lower contribution from CYP3A, other P450s (e.g., CYP2C9, CYP2D6, or CYP1A2) did not contribute to more than 17% of the etoricoxib 6′-methyl hydroxylase activity in microsomes of subject 24 (data not shown).

Testosterone 6β-hydroxylase activity (A) and inhibition of etoricoxib hydroxylase activity by troleandomycin (B) in a panel of human liver microsomes.

Troleandomycin (50 μM) was incubated with microsomal protein (1.0 mg/ml) as described in Fig. 4. Etoricoxib hydroxylase activity was assessed at a final etoricoxib concentration of 50 μM.

Immunoinhibition Studies with Native Human Liver Microsomes.

To confirm the P450 reaction phenotyping results obtained with chemical inhibitors and cDNA-expressed P450s, [14C]etoricoxib (50 μM) was incubated with native human liver microsomes in the absence and presence of immunoinhibitory anti-CYP3A4 peptide polyclonal antibodies. The anti-CYP3A4 antibody preparation has been shown to be highly selective for CYP3A4-catalyzed reactions in native human liver microsomes (Wang and Lu, 1997), and the results with etoricoxib indicated that the majority (∼60%) of the 6′-methyl hydroxylase activity was attributable to CYP3A4 (Fig. 6).

Effect of increasing concentrations of anti-CYP3A4 peptide or preimmune sera on the rate of etoricoxib hydroxylation in pooled native human liver microsomes.

The experiment was performed with a pool of microsomes, prepared by combining samples HHM-0228, HHM-0232, and HHM-0253.

Metabolism by cDNA-Expressed P450 Forms.

Of the P450 forms tested, CYP3A4 (20 pmol/h/pmol of P450), CYP2D6 (95 pmol/h/pmol of P450), and CYP2C19 (16 pmol/h/pmol of P450) exhibited high rates of hydroxylase activity (Table3). By comparison, the rate of hydroxylation in the presence of cDNA-expressed CYP1A2, CYP2A6, CYP2C8, CYP2C9, and CYP2E1 was low (≤2.3 pmol/h/pmol of P450). No activity was detected in (control) microsomes prepared from insect cells containing the selectable plasmid vector without cDNA insert (data not shown).

Metabolism of etoricoxib in the presence of insect cell microsomes containing various cDNA-expressed P450 proteins

To obtain meaningful information, the turnover rates obtained with the various cDNA-expressed P450 proteins were normalized with respect to the nominal abundance of each P450 protein in native human liver microsomes (Shimada et al., 1994; Lasker et al., 1998; Rodrigues, 1999). It is evident from the data presented in Table 3 that the hydroxylation of etoricoxib is largely mediated by CYP3A4 (∼60%), whereas a lesser contribution is made by other P450s, such as CYP2C9, CYP2D6, and CYP2C19. In agreement, etoricoxib hydroxylation catalyzed by insect cell microsomes containing recombinant CYP3A4 was characterized by an apparent Km (201 μM) similar to that obtained with native liver microsomes (Fig. 3). If theVmax (1.9 nmol/min/nmol of CYP3A4) is normalized with respect to the mean specific content of CYP3A4 (∼0.1 nmol/mg) in a bank of native human liver microsomes (Shimada et al., 1994), this yields a predicted Vmax of 0.19 nmol/min/mg in human liver microsomes. As shown in Table 1, this value is very close to the Vmax obtained with the pool of native human liver microsomes (sample HHM-0253,Vmax = 0.27 nmol/min/mg; 0.16 nmol/min/mg, when adjusted for the contribution of CYP3A, i.e.,Vmax · fm, CYP3A4 = 0.27 · 0.6).

Discussion

The results of these studies demonstrate that etoricoxib undergoes P450-dependent oxidation and that 6′-methyl hydroxylation represents the major metabolic pathway in NADPH-fortified human liver microsomes (Fig. 2A). By comparison, 1′-N-oxidation to M1 is a relatively minor pathway that agrees with previously reported data (Chauret et al., 2001). The C-oxidation of etoricoxib (to the 6′-carboxy metabolite M3) is the predominant pathway in vivo (Fig. 2B) and suggests that generation of M3 (retention time ∼10 min) in vitro requires the presence of cytosolic enzyme(s). In fact, M3 was shown to be formed by human liver S9 fraction in the presence of NAD+ (data not shown).

Because etoricoxib is extensively metabolized in humans (>90% of the dose), an attempt was made to predict the CLint,in vivo, based on estimates of apparentKm and Vmax in vitro (Table 1). The CLint, in vitro obtained with human liver microsomes was scaled (Obach et al., 1997) to yield a predicted CLint, in vivo of 3.1 (HHM-0253) to 9.7 (HHM-0228) ml/min/kg. These values compare favorably with the observed CLint, in vivo (8.3 ml/min/kg; mean ofn = 6 subjects; coefficient of variation = 25%; N. Agrawal unpublished results) calculated from the i.v. data (eq. 2).

In human liver microsomes, the 6′-methyl hydroxylation of etoricoxib was characterized as a relatively high apparentKm process (∼0.2 mM), a result that agrees well with earlier data (Chauret et al., 2001) and that has two clinical implications. First, etoricoxib would be expected to exhibit linear pharmacokinetics, which is in agreement with recent findings (N. Agrawal, unpublished observations). Second, one would expect etoricoxib (plasma Cmax in humans ≤3 μg/ml; ≤8 μM) to be a weak competitive inhibitor of hepatic CYP3A4.

Several lines of evidence (e.g., correlation analysis, P450 form selective inhibitors, and cDNA-expressed P450 proteins) have demonstrated that member(s) of the CYP3A subfamily, most likely CYP3A4, is (are) the principal human liver microsomal enzyme(s) involved in the hydroxylation of etoricoxib. In fact, data obtained with a bank of liver microsomes (n = 16 different organ donors) suggested that the contribution of CYP3A varied from 40% to 90% (mean ± S.D. = 68 ± 13.4%; Fig. 5), which was in agreement with the results obtained with a pool of human liver microsomes (Fig.4). However, it was not possible to evaluate the role of CYP3A5 (versus CYP3A4), because no attempt was made to measure the level of this enzyme in the microsomes used in this study, although the antipeptide antibody used for immunoinhibition studies has been shown to be selective (versus CYP3A5) for CYP3A4 (Wang and Lu, 1997).

Although the data point to a major role for CYP3A, results obtained with P450 form selective chemical inhibitors and recombinant proteins indicate that a number of other P450s (CYP2D6, CYP2C9, CYP1A2, and possibly CYP2C19) more or less equally contribute to the remainder (∼40%) of the 6′-methyl hydroxylase activity in native human liver microsomes. Even when the contribution of CYP3A is relatively low, as in subject 24 (Fig. 5), CYP2D6, CYP2C9, or CYP1A2 do not contribute to more than 17% of the metabolism of etoricoxib (data not shown).

For etoricoxib, the clinical relevance of the in vitro P450 reaction phenotype described herein will ultimately depend upon the fraction of the dose that is metabolized via 6′-methyl hydroxylation, which has been estimated to be ∼80% (based on urinary and fecal profiles of subjects dosed with [14C]etoricoxib; R. Halpin, unpublished observations). The results of this study indicate that CYP3A4 accounts for 40 to 90% of the total 6′-methyl hydroxylase activity in human liver microsomes, which implies that approximately 30 to 70% of the dose would be cleared by the enzyme (fm, CYP3A ≈ 0.3–0.7) in vivo. Therefore, it is possible that the area under the curve of etoricoxib would be relatively refractory to the effects of CYP3A4 inhibitors (≤3-fold increase) and inducers (≤60% decrease) (Rodrigues and Wong, 1997).

Interestingly, despite the fact that multiple P450 forms were shown to catalyze the 6′-methyl hydroxylation of etoricoxib (1–1300 μM), the data obtained with native human liver microsomes were adequately described by a single Km model (Fig. 3A). This finding indicates that CYP2C9, CYP2C19, CYP1A2, CYP2D6, and CYP3A4 are likely to be characterized by similar apparentKm values. As expected, CYP3A4 predominates by virtue of its high abundance in the liver (Shimada et al., 1994). Although CYP2C9, CYP2C19, and CYP2D6 are known to be polymorphically expressed (Bertilsson, 1995; Guengerich, 1995; Nasu et al., 1997; Ruas and Lechner, 1997), each enzyme plays a relatively minor role (≤10%) in the metabolism of etoricoxib. Therefore, the pharmacokinetic profile of etoricoxib is not expected to cosegregate with these polymorphisms.

In conclusion, etoricoxib is metabolized via 6′-methyl hydroxylation in human liver microsomes. The reaction is catalyzed by a number of P450 forms, although CYP3A4 accounts for the majority (40–90%) of the activity. The remainder of the activity is equally divided between a number of other P450s (e.g., CYP2D6, CYP2C9, CYP1A2, and possibly CYP2C19). In this regard, the P450 reaction phenotype of etoricoxib is unique and differs from that of other COX inhibitors.

(1996) The importance of nonspecific binding in in vitro matrices, its impact on enzyme kinetic studies of drug metabolism reactions, and implications for in vitro-in vivo correlations.Drug Metab Dispos24:1047–1049.